Overexpression of the human epidermal growth factor receptor 2 (HER2) gene product frequently associate with an aggressive clinical course, decreased disease-free survival, poor prognosis, and increased resistant and metastasis in human breast cancer. Therefore, anti-HER2 humanized mAb Herceptin has emerged as an anticancer agent for some form of breast cancer. However, our understanding the mechanisms through which HER2 influences sensitivity of breast cancer cells to anti-estrogenic and differentiation agents has been limited. This proposal is designed to delineate the mechanistic and functional significance of recently-identified novel targets of HER2 in breast cancer cells; to develop mechanism based pre-clinical models to evaluate whether Herceptin in combination with anti- hormonal or differentiation-inducing agents will have better antitumor activity; and to establish the molecular mechanism or mechanisms by which HER2 participates in the development of resistance against anti- hormonal and differentiation therapies. Our preliminary data suggest that HER2 overexpression in human breast cancer may be accompanied by an increased expression of G3BP (a Ras effector) and PPAR-gamma, and cytoplasmic localization of ER. We also show that Herceptin effectively blocked the phosphorylation and functions of G3BP, and also sensitizes breast cancer cells to PPAR ligand therapy. Our working hypothesis is that "HER2 deregulation stimulates the signaling pathways leading to enhanced cross-talk with ER and PPAR pathways, and consequently, to the development of resistance to anti-hormonal and differentiation therapies; these phenotypic changes can be suppressed or reversed by Herceptin." To address these hypotheses, our specific aims are to: (1) To determine the mechanism by which HER2 regulates the cytoplasmic localization of ER and MAPK activation in ER-positive breast cancer cells; (2) To examine the functional consequence of downregulation of HER2 signaling on the subcellular localization of ER and sensitivity to anti-hormonal agents; (3) To determine the mechanisms by which HER2 downregulation influences the responsiveness of breast cancer cells to PPAR-ligand therapy; and (4) To determine the expression characteristics and the prognostic value of HER2, G3BP, PPAR and subcellular localization of ER in patients with invasive breast cancer, and determine whether these molecular relationships correspond with clinical response to Herceptin by using archived specimens. Together, These studies will uniquely define the mechanisms through which HER2 and its downstream targets regulate survival and mitcgenesis in breast cancer cells, and will provide a novel rationale for therapy of breast cancer by combination of Herceptin with anti-hormonal or differentiation-inducing agents.